Heterogeneous fibroblasts contribute to fibrotic scar formation after spinal cord injury in mice and monkeys

Spinal cord injury (SCI) leads to fibrotic scar formation at the lesion site, yet the heterogeneity of fibrotic scar remains elusive. Here we show the heterogeneity in distribution, origin, and function of fibroblasts within fibrotic scars after SCI in mice and female monkeys. Utilizing lineage tracing and single-cell RNA sequencing (scRNA-seq), we found that perivascular fibroblasts (PFs), and meningeal fibroblasts (MFs), rather than pericytes/vascular smooth cells (vSMCs), primarily contribute to fibrotic scar in both transection and crush SCI. Crabp2 + /Emb+ fibroblasts (CE-F) derived from meninges primarily localize in the central region of fibrotic scars, demonstrating enhanced cholesterol synthesis and secretion of type I collagen and fibronectin. In contrast, perivascular/pial Lama1 + /Lama2+ fibroblasts (LA-F) are predominantly found at the periphery of the lesion, expressing laminin and type IV collagen and functionally involved in angiogenesis and lipid transport. These findings may provide a comprehensive understanding for remodeling heterogeneous fibrotic scars after SCI.

Dot plot showing that mouse and monkey CE-F express higher levels of collagen I and Fn1, while LA-F express higher levels of collagen IV and laminin.
Figure S1 PDGFRβ-CreER::R26-TdT transgenic mice are appropriate to track the cell fate of fibroblasts and pericytes/vSMCs.(A) Representative images showing that TdT+ cells (red) co-localize with PDGFRβ (green) in the uninjured spinal cord.Arrows indicate the colocalization of TdT and PDGFRβ.Scale bars: 50 μm.Left indicates the parenchyma, and the right indicates the meninges.(B) PDGFRβ-TdT+ cells (red) are located around vessels (arrows).Endothelial cells are labeled by Pdx (green).Three repeats are performed independently.Arrows indicate the adjacency of TdT and Pdx.Scale bars: 50 μm.(C) Representative images showing that TdT+ cells (red) co-localize with Sox9 (green, right) but not Sox10 (green, left) in the uninjured spinal cord.Arrows in the left images indicate no co-localization of TdT and Sox10, while arrows in the right images indicate the co-localization of TdT and Sox9.Scale bars: 50 μm.(D) The percentage of PDGFRβ+TdT+ and Sox9+TdT+ cells in TdT+ cells of the uninjured spinal cord.Data are shown as mean ± SEM. n = 3 mice per group.

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Figure S2 PDGFRβ+ cells proliferate and express Nestin after SCI and contribute to the formation of fibrotic scars.(A) Quantification of the percentage of proliferating PDGFRβ-TdT+ cells, as determined by co-expression of Ki67 and TdT.Data are shown as mean ± SEM. n = 3 mice per group.ns = 0.9340 (transection SCI vs. crush SCI at 14 dpi).ns = 0.9965 (transection SCI vs. crush SCI at 1 mpi).ns = 0.0591 (14 dpi vs. 1mpi in transection SCI), ns = 0.0783 (14 dpi vs. 1mpi in crush SCI).****P < 0.0001 by two-way ANOVA with Sidak's multiple comparisons test.(B-C) Representative images showing that in the transection (A) and crush (B) SCI models, PDGFRβ-TdT+ cells (red) contribute to the Nestin+ cells (green) in the lesion core at 5 dpi.b1-b2 and c1-c2, magnified boxed regions in B and

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Figure S3 Major cell types obtained from uninjured and transected mouse spinal cords.(A) PDGFRβ-TdT+ cells from uninjured and injured spinal cords were purified by FACS for single-cell RNA sequencing.(B) UMAP plots showing the identities of the major cell types obtained from uninjured and injured spinal cords at different time points.

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Figure S4 Pericytes/vSMCs are labeled in Myh11-CreER:R26-TdT mice.(A) Representative images showing the expression of TdT (red) and Desmin (green) in PDGFRβ-CreER::R26-TdT mice after transection or crush SCI.Dashed lines label the lesion sites.Scale bar: 250 μm.(B) The percentage of Desmin+TdT+ cells in TdT+ cells in the injury site of transection SCI and crush SCI model.Data are shown as mean ± SEM. n = 3 mice per group.ns, non-significant by two-sided, unpaired Student's t-test.P = 0.0593.(C) Representative image showing the co-localization of TdT (red) and CD13 (green) in Myh11-CreER::R26-TdT mice.Right panel showing the magnification image of boxed region of C. Scale bar: 250 μm, 50 μm (enlarged view).(D) Representative image showing the co-localization of TdT (red) and Desmin (green), as well as TdT (red) and αSMA (green) in Myh11-CreER::R26-TdT mice.Scale bar: 20 μm.

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Figure S8 Extended figures for gene expression in fibroblasts of mice.(A) Dot plot showing the expression of ECM related genes in fibroblasts identified in figure 3B, suggesting similarities between clusters 2 and 6. (B) Expression pattern of marker genes of pericytes/vSMCs, PFs and MFs.(C) Dividing fibroblasts were extracted and reclustered in new UMAP coordinates.